Mice completely lacking immunoproteasomes show major changes in antigen presentation.

The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes had not been available. Having now developed mice lacking the three immunoproteasome catalytic subunits, we found that the dendritic cells of these mice had defects in presenting several major histocompatibility complex (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes was markedly reduced in immunoproteasome-deficient animals compared with wild-type animals, whereas presentation of MHC class II peptides was unaffected. According to mass spectrometry, the repertoire of MHC class I-presented peptides was ∼50% different from that in wild-type mice, and these differences were sufficient to stimulate robust transplant rejection of wild-type cells in mutant mice. These results indicated that immunoproteasomes were more important in antigen presentation than previously thought.

Development of UHPLC/Q-TOF Analysis Method to Screen Glycerin for Direct Detection of Process Contaminants 3-Monochloropropane-1,2-diol Esters (3-MCPDEs) and Glycidyl Esters (GEs).

The U.S. Food and Drug Administration's (FDA's) Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats since 2007. Renal failure accounted for 30% of reported cases. Jerky pet treats contain glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Glycidyl esters (GEs) and 3-monochloropropanediol esters (3-MCPDEs) are food contaminants that can form in glycerin during the refining process. 3-MCPDEs and GEs pose food safety concerns, as they can release free 3-MCPD and glycidol in vivo. Evidence from studies in animals shows that 3-MCPDEs are potential toxins with kidneys as their main target. As renal failure accounted for 30% of reported pet illnesses after the consumption of jerky pet treats containing glycerin, there is a need to develop a screening method to detect 3-MCPDEs and GEs in glycerin. We describe the development of an ultra-high-pressure liquid chromatography/quadrupole time-of-flight (UHPLC/Q-TOF) method for screening glycerin for MCPDEs and GEs. Glycerin was extracted and directly analyzed without a solid-phase extraction procedure. An exact mass database, developed in-house, of MCPDEs and GEs formed with common fatty acids was used in the screening.

Peptidomic analysis of type 1 diabetes associated HLA-DQ molecules and the impact of HLA-DM on peptide repertoire editing.

HLA-DM and class II associated invariant chain (Ii) are key cofactors in the MHC class II (MHCII) antigen processing pathway. We used tandem mass spectrometry sequencing to directly interrogate the global impact of DM and Ii on the repertoire of MHCII-bound peptides in human embryonic kidney 293T cells expressing HLA-DQ molecules in the absence or presence of these cofactors. We found that Ii and DM have a major impact on the repertoire of peptides presented by DQ1 and DQ6, with the caveat that this technology is not quantitative. The peptide repertoires of type 1 diabetes (T1D) associated DQ8, DQ2, and DQ8/2 are altered to a lesser degree by DM expression, and these molecules share overlapping features in their peptide binding motifs that are distinct from control DQ1 and DQ6 molecules. Peptides were categorized into DM-resistant, DM-dependent, or DM-sensitive groups based on the mass spectrometry data, and representative peptides were tested in competitive binding assays and peptide dissociation rate experiments with soluble DQ6. Our data support the conclusion that high intrinsic stability of DQ-peptide complexes is necessary but not sufficient to confer resistance to DM editing, and provide candidate parameters that may be useful in predicting the sensitivity of T-cell epitopes to DM editing.

Type 1 diabetes associated HLA-DQ2 and DQ8 molecules are relatively resistant to HLA-DM mediated release of invariant chain-derived CLIP peptides.

HLA-DM is essential for editing peptides bound to MHC class II, thus influencing the repertoire of peptides mediating selection and activation of CD4(+) T cells. Individuals expressing HLA-DQ2 or DQ8, and DQ2/8 trans-dimers, have elevated risk for type 1 diabetes (T1D). Cells coexpressing DM with these DQ molecules were observed to express elevated levels of CLIP (Class II associated invariant chain peptide). Relative resistance to DM-mediated editing of CLIP was further confirmed by HPLC-MS/MS analysis of eluted peptides, which also demonstrated peptides from known T1D-associated autoantigens, including a shared epitope from ZnT8 that is presented by all four major T1D-susceptible DQ molecules. Assays with purified recombinant soluble proteins confirmed that DQ2-CLIP complexes are highly resistant to DM editing, whereas DQ8-CLIP is partially sensitive to DM, but with an apparent reduction in catalytic potency. DM sensitivity was enhanced in mutant DQ8 molecules with disruption of hydrogen bonds that stabilize DQ8 near the DM-binding region. Our findings show that T1D-susceptible DQ2 and DQ8 share significant resistance to DM editing, compared with control DQ molecules. The relative resistance of the T1D-susceptible DQ molecules to DM editing and preferential presentation of T1D-associated autoantigenic peptides may contribute to the pathogenesis of T1D.

Expression of the mouse MHC class Ib H2-T11 gene product, a paralog of H2-T23 (Qa-1) with shared peptide-binding specificity.

The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with the highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted α3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP(+) and TAP(-) cells. Soluble T11D3 was generated by folding in vitro with Qa-1 determinant modifier, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qa-1 determinant modifier peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1-restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus, T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function.

ERAAP and tapasin independently edit the amino and carboxyl termini of MHC class I peptides.

Effective CD8(+) T cell responses depend on presentation of a stable peptide repertoire by MHC class I (MHC I) molecules on the cell surface. The overall quality of peptide-MHC I complexes (pMHC I) is determined by poorly understood mechanisms that generate and load peptides with appropriate consensus motifs onto MHC I. In this article, we show that both tapasin (Tpn), a key component of the peptide loading complex, and the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP) are quintessential editors of distinct structural features of the peptide repertoire. We carried out reciprocal immunization of wild-type mice with cells from Tpn- or ERAAP-deficient mice. Specificity analysis of T cell responses showed that absence of Tpn or ERAAP independently altered the peptide repertoire by causing loss as well as gain of new pMHC I. Changes in amino acid sequences of MHC-bound peptides revealed that ERAAP and Tpn, respectively, defined the characteristic amino and carboxy termini of canonical MHC I peptides. Thus, the optimal pMHC I repertoire is produced by two distinct peptide editing steps in the endoplasmic reticulum.

Endoplasmic reticulum aminopeptidase associated with antigen processing defines the composition and structure of MHC class I peptide repertoire in normal and virus-infected cells.

The MHC class I (MHC-I) molecules ferry a cargo of peptides to the cell surface as potential ligands for CD8(+) cytotoxic T cells. For nearly 20 years, the cargo has been described as a collection of short 8-9 mer peptides, whose length and sequences were believed to be primarily determined by the peptide-binding groove of MHC-I molecules. Yet the mechanisms for producing peptides of such optimal length and composition have remained unclear. In this study, using mass spectrometry, we determined the amino acid sequences of a large number of naturally processed peptides in mice lacking the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP). We find that ERAAP-deficiency changed the oeuvre and caused a marked increase in the length of peptides normally presented by MHC-I. Furthermore, we observed similar changes in the length of viral peptides recognized by CD8(+) T cells in mouse CMV-infected ERAAP-deficient mice. In these mice, a distinct CD8(+) T cell population was elicited with specificity for an N-terminally extended epitope. Thus, the characteristic length, as well as the composition of MHC-I peptide cargo, is determined not only by the MHC-I peptide-binding groove but also by ERAAP proteolysis in the endoplasmic reticulum.

Utility of characteristic QTOF MS/MS fragmentation for MHC class I peptides.

Systematic investigation of cellular process by mass spectrometric detection of peptides obtained from proteins digestion or directly from immuno-purification can be a powerful tool when used appropriately. The true sequence of these peptides is defined by the interpretation of spectral data using a variety of available algorithms. However peptide match algorithm scoring is typically based on some, but not all, of the mechanisms of peptide fragmentation. Although algorithm rules for soft ionization techniques generally fit very well to tryptic peptides, manual validation of spectra is often required for endogenous peptides such as MHC class I molecules where traditional trypsin digest techniques are not used. This study summarizes data mining and manual validation of hundreds of peptide sequences from MHC class I molecules in publically available data files. We herein describe several important features to improve and quantify manual validation for these endogenous peptides--post automated algorithm searching. Important fragmentation patterns are discussed for the studied MHC Class I peptides. These findings lead to practical rules that are helpful when performing manual validation. Furthermore, these observations may be useful to improve current peptide search algorithms or development of novel software tools.

Large scale mass spectrometric profiling of peptides eluted from HLA molecules reveals N-terminal-extended peptide motifs.

The majority of >2000 HLA class I molecules can be clustered according to overlapping peptide binding specificities or motifs recognized by CD8(+) T cells. HLA class I motifs are classified based on the specificity of residues located in the P2 and the C-terminal positions of the peptide. However, it has been suggested that other positions might be relevant for peptide binding to HLA class I molecules and therefore be used for further characterization of HLA class I motifs. In this study we performed large-scale sequencing of endogenous peptides eluted from K562 cells (HLA class I null) made to express a single HLA molecule from HLA-B*3501, -B*3502, -B*3503, -B*3504, -B*3506, or -B*3508. Using sequence data from >1,000 peptides, we characterized novel peptide motifs that include dominant anchor residues extending to all positions in the peptide. The length distribution of HLA-B35-bound peptides included peptides of up to 15 residues. Remarkably, we determined that some peptides longer than 11 residues represented N-terminal-extended peptides containing an appropriate HLA-B35 peptide motif. These results provide evidence for the occurrence of endogenous N-terminal-extended peptide-HLA class I configurations. In addition, these results expand the knowledge about the identity of anchor positions in HLA class I-associated peptides that can be used for characterization of HLA class I motifs.

Identification of naturally processed ligands in the C57BL/6 mouse using large-scale mass spectrometric peptide sequencing and bioinformatics prediction.

Most major histocompatibility complex (MHC) class I-peptide-binding motifs are currently defined on the basis of quantitative in vitro MHC-peptide-binding assays. This information is used to develop bioinformatics-based tools to predict the binding of peptides to MHC class I molecules. To date few studies have analyzed the performance of these bioinformatics tools to predict the binding of peptides determined by sequencing of naturally processed peptides eluted directly from MHC class I molecules. In this study, we performed large-scale sequencing of endogenous peptides eluted from H2K(b) and H2D(b) molecules expressed in spleens of C57BL/6 mice. Using sequence data from 281 peptides, we identified novel preferred anchor residues located in H2K(b) and H2D(b)-associated peptides that refine our knowledge of these H2 class I peptide-binding motifs. The analysis comparing the performance of three bioinformatics methods to predict the binding of these peptides, including artificial neural network, stabilized matrix method, and average relative binding, revealed that 61% to 94% of peptides eluted from H2K(b) and H2D(b) molecules were correctly classified as binders by the three algorithms. These results suggest that bioinformatics tools are reliable and efficient methods for binding prediction of naturally processed MHC class I ligands.

High Sensitivity Measurement of Pancreatic Polypeptide and Its Variant in Serum and Plasma by LC-MS/MS.

Aliquots of serum or plasma samples are combined with stable isotope labeled internal standard. Pancreatic polypeptide (PP) and its truncated variant PP3-36 are enriched by incubation with anti-PP antibody conjugated to magnetic beads. Peptides are eluted from beads in acidic buffer and the samples analyzed using liquid chromatography coupled with tandem mass spectrometry. Instrumental analysis of PP and PP3-36 is performed using electrospray ionization ESI in positive ion mode and multiple reaction monitoring (MRM) acquisition.

Caracterización socioeconómica de fincas cafeteras del departamento de Nariño, Colombia / Socioeconomic characterization of coffee farms of the department of Nariño, Colombia

RESUMEN La adopción de tecnologías generadas para el cultivo del café depende, en gran medida de factores, como la disponibilidad de recursos y el nivel de escolaridad de los productores, lo cual, determina la importancia de analizar las condiciones socioeconómicas, en la estructuración de los sistemas productivos de café. El objetivo de esta investigación fue analizar las principales características sociales y económicas de los cafeteros en los ecotopos 220A y 221A, departamento de Nariño. Con base en un marco muestral de 16.767 predios cafeteros, se seleccionaron aleatoriamente 159 productores (86, del ecotopo 220A y 73, del 221A), con el objeto de aplicar el formulario de encuesta. En el análisis estadístico, 58 variables categóricas fueron sometidas a un análisis multivariado, mediante el método de correspondencias múltiples y análisis de clasificación jerárquica. El ecotopo 220A, se caracterizó por tener áreas de café entre 1 y 3 hectáreas, viviendas con techos de eternit y zinc, pisos de cemento, energía eléctrica, acueducto y unidad sanitaria. El rendimiento está entre 1.001 y 2.000kg.ha-1, costos de producción menores a COP1.500.000 (USD444,83). Los caficultores asisten a jornadas de capacitación y su núcleo familiar está compuesto por 4-7 personas. En el ecotopo 221A predominan las casas con techo de teja, pisos en cemento, energía eléctrica, acueducto y unidad sanitaria; las aguas servidas se disponen en pozos sépticos. No se aplican las buenas prácticas agrícolas (BPA), los rendimientos son menores a 1.500kg.ha-1 cps (café pergamino seco) y los costos de producción son inferiores a COP1.500.000ha-1. año-1 (USD444,83).

ABSTRACT The adoption of technologies generated for the coffee crop, depends to a great degree on factors such as the availability of resources and the producer´s scholarship which determines the importance of socioeconomic conditions in the coffee productive system structure. The objective of this investigation was to analyze the main coffee grower´s social and economic characteristics in the 220A and 221A Nariño Department ecotopes. 159 producers (86 from the ecotype 220A and 73 from the ecotope 221A), were selected randomly based on a sampling frame of 16.767 coffee farms, with the purpose of applying the survey questionnaire. In the statistical analysis, 58 categorical variables were used, they were subjected to a multivariate analysis using the multiple correspondence method and hierarchical classification analysis. The 220A ecotope was characterized by having coffee areas between 1 and 3 hectares, houses with eternit and zinc roofs, cement floors, electric power, aqueduct and sanitary unit. The performance is between 1.001 and 2.000kg.ha1, the cost of production is less than COP1.500,000 (USD44,83); the coffee growers attend training sessions and their family nucleus is made up of 4 - 7 people. In the ecotope 221A the houses with tile roof, cement floors, electricity, aqueduct and sanitary unit predominate; wastewater is disposed of in septic tanks. Good agricultural practices (GAP) aren't used, the yields are less than 1.500kg.ha-1 (dried parchment coffee) and the cost of production is less than $1.500.000 ha-1. year-1

Evaluación del rendimiento y calidad del fruto de parentales e híbridos de lulo de Castilla / Evaluation of the yield and fruit quality of parental and hybrids of lulo de Castilla

RESUMEN Solanum quitoense es una planta de gran relevancia para emprender proyectos productivos con fines de exportación, como frutal exótico o para la industria. Su importancia radica en la posibilidad de aportar al desarrollo de los productores de la región andina, debido a que el lulo es demandado en el mercado por su sabor, aroma, propiedades nutritivas y organolépticas. A pesar de su importancia, esta especie presenta deficiencias tecnológicas, entre las cuales, se destaca la falta de cultivares mejorados, que permitan garantizar mayores rendimientos y calidad de fruta y establecer su eficiencia agronómica, a través de diferentes ambientes. El objetivo de este trabajo fue evaluar el comportamiento del rendimiento y de las variables relacionadas con la fruta, en poblaciones de lulo de Castilla. Se utilizaron ocho parentales y 10 híbridos. En los municipios de La Florida y Buesaco, ubicados en el departamento de Nariño, se establecieron dos ensayos, bajo un diseño de Bloques Completos al Azar, con tres repeticiones. La interacción genotipo por ambiente fue significativa para el peso de fruto (PF), diámetro ecuatorial, sólidos solubles totales, contenido de jugo y rendimiento (RTO). En La Florida, B1, B2, B3, B4xB5 y B2XLaSelva fueron los de mejor comportamiento en cuanto a RTO, con promedios entre 6,64 a 9,35t.ha-1 y PF, con 143 a 167g, en su orden. En Buesaco, se destacaron B1 y B2xB8 con RTOs de 7,72 y 9,43t.ha-1 y PF, entre 92,03 y 112,97g, promedios que están por encima del promedio regional y son la base para mejorar estas características.

ABSTRACT Solanum quitoense is a plant of great relevance to undertake productive projects for export as an exotic fruit or for industry. Its importance lies in the possibility of contributing to the development of producers in the Andean region, because the lulo is demanded in the market for its flavor, aroma, nutritional and organoleptic properties. Despite its importance, this species has technological deficiencies, among which, the lack of improved cultivars that guarantee greater yields and fruit quality and establish its agronomic efficiency through different environments is highlighted. The objective of this work was to evaluate the performance of the yield and traits related to the fruit in populations of lulo de Castilla. Eight parents and 10 hybrids were used. In the municipalities of La Florida and Buesaco located in the department of Nariño, two trials were established under a Randomized Complete Blocks design with three repetitions. Genotype interaction by environment was significant for fruit weight (FP), equatorial diameter, total soluble solids, juice content and yield (RTO). In La Florida, B1, B2, B3, B4xB5 and B2XLaSelva were the best performers in terms of RTO with averages between 6.64 to 9.35t.ha-1 and PF with 143 to 167g, in order. In Buesaco, B1 and B2xB8 stood out with RTOs of 7.72 and 9.43t.ha-1, PF between 92.03 and 112.97g, averages that are above the regional average and are the basis for improving these characteristics.

Regulation of HLA-DR peptide occupancy by histone deacetylase inhibitors.

Numerous molecular effects have been attributed to histone deacetylase inhibitors (HDACI's), including the induction of major histocompatibility (MHC) genes. Here we report that one FDA approved HDACI, Vorinostat, and a second HDACI currently in clinical trials, Entinostat, reduce the ratio of class II associated invariant peptide (CLIP) to the MHC class II molecule, HLA-DR, indicating an increase in the non-CLIP peptides bound to HLA-DR. The HDACI effects are apparent with immortalized B-cells, HLA-DR constitutive melanoma cells and with melanoma cells expressing HLA-DR due to transformation with an expression vector for the HLA-DR gene co-activator, CIITA. Entinostat treatment leads to upregulation of Cathepsin L1, and the HLA-DR peptidome of the Entinostat treated cells is consistent with increased Cathepsin L1 mediated proteolysis. These results indicate that HDACI treatments may alter the HLA-DR peptidome of cells in patients and provide a way to identify novel immunogens for vaccinations and the study of autoantigens.